The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to elucidate the mechanism of action of the phorbol esters and their endogenous analog, the lipophilic second messenger sn-1,2-diacylglycerol. Protein kinase C (PKC) is the major receptor for these compounds, and our emphasis is correspondingly directed at his family of isozymes. In a collaborative effort with the groups of Victor Marquez, NCI, and Shaomeng Wang, Georgetown University, we combine mutational analysis and pharmacological characterization with computer modeling and chemical synthesis to probe ligand - PKC interactions. One current direction is the analysis of the interaction of hydrophobic residues in the C1 domain of PKC with both ligand and with the lipid bilayer. Different residues contribute differentially to these two interactions, and the contributions depend, moreover, on the specific ligand. For example, diacylglycerol shows markedly greater dependence on W22 in the C1 domain than do the phorbol esters. Our emerging insights contribute to ligand design, and we now have synthetic ligands with nanomolar affinity and with unique selectivity between classes of diacylglycerol receptors. Other studies seek to characterize novel classes of PKC modulators identified from database analysis. Localization of PKC potentially plays an important role in determining its specificity. We have found that different ligands with different biological effects cause PKC delta to distribute to different intracellular locations. On-going analysis indicates that lipophilicity is one factor contributing to this differential localization. Whereas relatively hydrophilic derivatives position PKC delta at the nuclear membrane, those of intermediate hydrophobicity direct PKC initially to the plasma membrane. Although PKC is the major receptor for the phorbol esters, several novel families of proteins with C1 domains have now been identified which also recognize phorbol esters with high affinity. The RasGRP family members, activators of low molecular weight GTPases such as Ras and Rap, are of particular interest. Phorbol esters bind RasGRP1 and RasGRP3 with similar affinity to PKC, induce translocation in intact cells, and lead to activation of Ras and of Erk1/2, downstream targets of Ras. Localization of RasGRP3 in response to ligands depends on specific concentration. Whereas lower concentrations cause plasma membrane localization, higher concentrations cause localization to the nuclear membrane and perinuclear region. Since localization drives access to its targets, such differential localization may control RasGRP3 specificity. The phorbol related diterpene resiniferatoxin acts as an ultrapotent analog of capsaicin and has permitted characterization of specific capsaicin (vanilloid) receptors. Its differential ability to desensitize vanilloid receptors makes it an attractive therapeutic candidate for treatment of chronic pain and other conditions in which vanilloid sensitive pathways are involved (e.g. urge incontinence). Structure-function studies of the cloned vanilloid receptor, VR1, are beginning to reveal the regions of the receptor involved in ligand recognition. Cross-linking indicates that the receptor exists as a multimer; the influence of oligomer composition on receptor properties is currently under investigation. Using strategies developed for constrained diacylglycerols, we and our collaborator (J. Lee, Seoul National University) have designed high affinity agonists orders of magnitude more potent than capsaicin. We have also developed multiple classes of competitive antagonists. On-going work seeks to further characterize their properties.